Amplification of specific nucleic acid sequences via polymerase chain reaction (PCR) is a mature technique and a powerful tool in medical and biological researching. Three major steps, “denaturation,” “annealing” and “extension,” each requiring different reaction temperatures, are necessary in this biochemical process. In today's commercialized PCR amplification technology, a sample is prepared to contain a template DNA to be amplified, a pair of oligonucleotide primers complementary to a specific sequence of each single strand of the template DNA, a thermostable DNA polymerase, and deoxynucleotide triphosphates (dNTP). A specific portion of the nucleic acid sequence of the template DNA is then amplified by repeatedly heating and cooling the sample so that the sample is cycled through three different temperatures.
The first step in PCR is the denaturation step, in which the sample is heated to a high temperature so that the double-stranded template DNA is separated into single-stranded DNAs. The second step is the annealing step, in which the sample is cooled to a lower temperature so that the primers can bind to the single-stranded DNAs formed in the first step, forming DNA-primer complexes. The last step is the polymerization (extension) step, in which the sample is maintained at a suitable temperature and the primers in the DNA-primer complexes are extended by the action of the DNA polymerase, thereby generating new single-stranded DNAs that are complementary to each strand of the template DNA. In each cycle consisting of the above three steps, the DNA sequences between the binding sites of the two primers are replicated. Typically, millions of the target nucleic acid sequence copies can be produced by repeating the PCR cycle which comprises the three steps of denaturation, annealing and extension, at different temperatures respectively for about 20 to 40 times.
The temperature of the denaturation step typically ranges from 90 to 94° C. The temperature of the annealing step is selected according to the melting temperatures (Tm) of the primers used, which typically ranges from 35 to 65° C. The typical temperature for the polymerization step is 72° C., since the most frequently used DNA polymerase, Taq DNA polymerase (a thermostable DNA polymerase extracted from Thermus aquaticus), has optimal activity at that temperature. Since Taq DNA polymerase has a broad range of temperature, a two-step temperature cycle can also be used, in which the polymerization temperature is almost the same as the annealing temperature.
In a conventional commercial PCR machine (i.e., a thermocycler), the temperature of the sample is controlled by a thermal conduction. Briefly, a reaction vessel containing the PCR sample is made in contact with a solid metal block having a high thermal conductivity. The metal block is connected to heating and cooling devices so that its temperature can be changed to achieve desired temperatures. The conventional PCR machine adopting such method often uses a gold-plated silver block that has very high thermal conductivity and/or the Peltier cooling method in order to achieve rapid temperature changes. However, conventional PCR thermal cycling is an inefficient process because it requires the heating and cooling of material other than the PCR sample itself, which takes additional time and energy. In addition, thermocyclers are generally expensive due to the delicate nature of the machine.
Convective PCR methods were developed to perform PCR on an apparatus with two temperature-controlled devices (Krishnan, M. et al., 2002, Science 298: 793). Benett et al., disclosed the methods and apparatuses for convective PCR (CPCR), wherein the convectively-driven sample solution circulates in a sealed O-shaped chamber heated at one side in U.S. Pat. No. 6,586,233, issued on Jul. 1, 2003. Hwang et al. disclosed the methods and apparatuses for convective PCR, wherein multiple heat sources are used to maintain different temperature zones in the sample solution so that the three steps of PCR occur sequentially and repeatedly while the sample cycles between each zone in U.S. patent application Ser. No. 10/801,342, published on Mar. 15, 2004 under US Publication No. 2004/0152122.
Although some methods have been provided for a convective PCR, for which the apparatuses were involved complicated structures, and were expensive, therefore hindering their commercial application. There is still a need for a more convenient and practical method and apparatus for a convective PCR (CPCR).